Adaptive osmoregulation in successive flushes of Agaricus bisporus enhances nutritional value of second flush mushrooms via elevated protein and free amino acid production

Agaricus bisporus, a widely cultivated edible button mushroom, is grown on composted substrates in successive flushes. While the first flush exhibits highest yields, later flushes exhibit reduced productivity. These differences are due to physico-chemical changes of the substrate: e.g. nutrient and water availability. Mushroom growth relies on maintaining an osmotic gradient between the substrate and fruiting body to allow resource translocation to the mushrooms. Classically, mannitol is known as the most important osmolyte in A. bisporus mushrooms. In this study, we investigated osmotic regulation during mushroom development over two flushes. We found that under standard cultivation conditions, the profile of organic osmolytes varies significantly between flushes in response to a changing substrate composition. During the first flush, when carbon is still abundant, mannitol is the dominant organic osmolyte supporting fruiting body expansion. In contrast, the second flush occurs under carbon-limited conditions, prompting a shift in osmotic strategy: fruiting bodies accumulate more nitrogen, with free amino acids contributing substantially to osmotic potential, reaching up to 11% of the dry weight, thereby enhancing nutritional value. This is one of the highest levels found in unprocessed food. These findings reveal a dynamic osmotic adaptation mechanism in A. bisporus, where different osmolytes are utilized depending on substrate nutrient availability. This metabolic flexibility likely helps sustaining growth under suboptimal conditions and results in mushrooms with improved nutritional quality in later flushes. Understanding the interplay between substrate composition, osmolyte profiles, and fruiting body physiology offers new avenues for optimizing mushroom yield and quality in commercial systems.